Analysis of fsll3 5 conditionally immortal cell lin es.
5.1: Introduction.
The conditionally immortalised cell lines originally isolated by Parmjit Jat (Jat and Sharp, 1989) following the infection of rat embryo fibroblasts with a retrovirus transducing the temperature sensitive large T antigen mutant tsA5^ growth arrested irreversibly after inactivation of the T antigen at the non-permissive temperature for 72 hours or more. Following growth arrest at the non-permissive temperature these cell lines are unable to grow even if the stability of the tsA5S T antigen is restored by shifting the cells back to the permissive temperature. This growth arrest occurs at either the G1 or G2 phase of the cell cycle and it is believed that these cells are recapitulating senescence (E.S.Gonos et al, in preparation). When these cell lines are shifted to the non-permissive temperature they remain metabolically active and do not show any degree of cell death. In contrast, Zheng et at. (1994) have reported that REFs similarly immortalised with the tsA5S T antigen mutant not only growth arrest at the non- permissive temperature but also undergo apoptosis. They have suggested that this is due to the dissociation of T antigen-p53 complexes thereby releasing p53.
The cell lines which I isolated at the permissive temperature following the cotransfection of fsll3 5 with either 5041 or 5031 were also conditionally immortal. In these cell lines, however, the majority of the functions carried by T antigen are retained upon shift up to the non-permissive temperature, since only one of the cooperating T antigen mutants carries the tsA5S point mutation and is thermolabile. Only the T antigen function(s) being provided exclusively by the f5ll35 molecule are inactivated at the non-permissive temperature. The functional domain provided by the f jll3 5 molecule, which is disrupted in the 5041 and 5031 molecules, is the domain required for T antigen's association with p53. It is possible, however, that this domain may also provide other functions necessary for T antigen's growth stimulatory effects. The existence of another activity in this domain has been suggested by Cavender et al. (1995) who demonstrated that a T antigen molecule with a mutation in this domain.
Chapter 5._________________________________________________ 142
which was still able to associate with p53, was unable to transform in cooperation with an activated ras oncogene. It is possible that the continued presence of the functions provided by the 5041 or 5031 proteins in my conditionally immortal cell lines when their growth is arrested at the non-permissive temperature, may change the characteristics of the growth arrest compared to that observed in the tsASS cell lines. It may be at that the functions provided by the 5041 or 5031 proteins prevent irreversible growth arrest in these cells and they may be able to resume growth upon being returned to the permissive temperature.
In this chapter I descibe two lines of analysis which I carried out to further characterise the conditionally immortal ^51135+5041/5031 cell lines. Firstly, I carried out two proliferation assays on six of these cell lines to determine whether the growth arrest in these cell lines, upon shift up to the non-permissive temperature, is reversible. Secondly, I have attempted to overcome the growth arrest in these six cell lines by re introducing expression of either wild type T antigen or the non-temperature sensitive d ll\3 5 mutant at the non-permissive temperature.
5.2: Results.
5.2.1: The growth arrest in the f^ll35 plus 5041 or 5031 cell lines is irreversible. The ability of representative conditionally immortal cell lines to grow at either the permissive temperature (33.0°C), the non-permissive temperature (39.5°C) or the permissive temperature following growth arrest at the non-permissive temperature for 7 days (39.5°C to 33.0°C) was determined by two growth assays. The first involved plating 10,000 cells on multiple plates and counting the resulting number of cells per plate, at several time points, after incubation of the plates under the three conditions. This cell number assay yielded data for the whole population of cells initially plated, however, if a sub-population of these cells retained the ability to grow upon being returned to the permissive temperature, then the results would become distorted. This was overcome in the second assay, where 1000 cells were plated on six dishes, and two of each were incubated under each condition. After at least 14 days at their final temperature the resulting colonies were stained and counted. With this colony assay the viability of each cell plated could be determined by its ability to form
an
individual colony. From the results of the colony assays it was possible to determine whether the majority of the population of cells for each cell line were irreversibly arresting and only a sub-population attained the ability to overcome the growth arrest, or whether the growth arrest was reversible for the majority of the population.The results of these growth assays are shown in Figures 5.1-6. In each figure the left graph represents the cell number assays, where the number of cells obtained under
each condition is plotted against the number of days. Representative plates were shifted up to 39.5°C on day 1 (and first counted at 39.5°C on day 4) and returned to 33.0°C on day 7 (39.5°C to 33.0°C cells were first counted on day 10). The graphs on the right side of each figure show the results for the colony assays presented as number of colonies obtained for each of the three growth conditions.
In total six tsA5S conditionally immortal cell lines were assayed for their ability to grow following growth arrest at 39.5°C. Four of these were isolated from my pUC19fjA58 transfections while the other two (tsaS and tsal4) are two of the cell lines originally isolated by Jat and Sharp (1989) using the pZiprjA58 retrovirus; tsal4 is the cell line I used to assay the maintenance of immortalisation by T antigen mutants (Chapter 3). Figure 5.1 A and B show the results obtained with the tsa8 and tsal4 cell lines respectively. The majority of the cells appeared to irreversibly growth arrest when shifted to the non-permissive temperature, however a proportion of the cells in each case appeared to be able to resume growth at the permissive temperature. The proportion of cells able to overcome the growth arrest and resume growth at 33.0°C appeared to be greater in the case of tsal4 (as indicated by the colony assay). tsASS cell lines 2 and 8 (Fig. 5.1C & 5.2A respectively) gave similar results with the colony assays. However, it seems that neither of these two cell lines had successfully arrested following 7 days at 39.5°C, since the number of cells obtained after 7 days was similar at either temperature. A reduction in cell number at 39.5°C was only observed after 14 days in either cell line. Thus in these assays the cells were returned to the permissive temperature before the majority of cells had ceased dividing. tsASS cell lines 16 and 17 (Fig. 5.2B & C respectively) both arrested rapidly at 39.5°C. In cell line 17, little growth occurred upon shifting back to 33.0°C from 39.5°C, while in cell line 16 some of the cells were able to overcome the growth arrest to eventually yield a similar number of cells as obtained at 33.0°C. In each case, the fact that only a proportion of the cells overcome the growth arrest when returned to the permissive temperature is indicated by the colony assays.
The results obtained for two cell lines immortalised with each non-temperature sensitive combination are shown in Figure 5.3 (J/1135-H5041 cell lines) and Figure 5.4 (<7/1135+5031 cell lines). These four cell lines are not conditionally immortal and therefore did not arrest at either temperature. The numbers of colonies obtained were similar on plates incubated under the three growth conditions for all four cell lines. The cell number assays for all four cell lines indicated that non-conditional cell lines generally yielded 5-10 fold more cells at 39.5°C than at 33.0°C after 14 days growth.
Figure 5.5 shows the results obtained with three 1135+5041 cell lines while Figure 5.6 shows the results for three fjl 135+5031 cell lines. The results obtained for each of these cell lines were similar. The majority of the cells irreversibly growth
Chapter 5,
144
Figure 5.1: Ability o f tsASH cell lines to grow at the permissive temperature following growth arrest at the non-permissive temperature: 1.
A. tsaS (tsASS) 10? 10® -
i
s Days B. tsal4 (tsASS) 10® 10® 10® DaysC. tsASS cell line 2
Growth conditions m cn o CO Growth conditions 300 - % 200 - 100 - Days ^ y m o U m Growth conditions
The indicated cell lines were assayed for their ability to grow at the permissive temperature after growth arrest resulting from incubation at the non-permissive temperature for 7 days. The line graphs indicate the number of cells obtained on day 1, day 4, day 7, day 10, day 14, day 17 and day 21 following the incubation of the cells (10,000 seeded per 10cm plate) at either the permissive temperature
(33.0°C; — o— ), the non-permissive temperature (39.5°C; «■— ) or cells which were shifted (the day after plating) to the non-permissive temperature for 7 days then shifted back to the permissive temper ature ( - --0-- ). The bar graphs represent the number of colonies obtained following the incubation of
the same cell lines (1000 cells seeded per 6cm plate) after incubation at either the permissive temperature (33°C), the non-permissive temperature (39°C) or that were shifted (the day after plating) to the non- permissive temperature for 7 days then shifted back to the permissive temperature (39°C to 33°C). Colonies were stained after two weeks (33.0°C plates) or three weeks (39.5°C and 39.5°C to 33.0°C plates) with methylene blue and counted.
Chapter 5.
Figure 5.2: Ability of tsASS cell lines to grow at the permissive temperature following growth arrest at the non-permissive temperature: 2.
A. tsASS cell line 8 10®
105
1Q3
Days
B. tsASS cell line 16
8
z
Days
C. tsASS cell line 17
I
300 - 00 - y ^ au m 0\ U ^ m m o m Growth conditions y y ay m U m m o m Growth conditions Days Growth conditionsThe indicated cell lines were assayed for their abihty to grow at the permissive temperature after growth arrest resulting from incubation at the non-permissive temperature for 7 days. The line graphs indicate the number of cells obtained on day 1, day 4, day 7, day 10, day 14, day 17 and day 21 following the incubation of the cells (10,000 seeded per 10cm plate) at either the permissive temperature
(33.0°C; — o— ), the non-permissive temperature (39.5°C; » ) or cells which were shifted (the day
after plating) to the non-permissive temperature for 7 days then shifted back to the permissive temper ature ( - --0-- ). The bar graphs represent the number of colonies obtained following the incubation of
the same cell lines (1000 cells seeded per 6cm plate) after incubation at either the permissive temperature (33°C), the non-permissive temperature (39°C) or that were shifted (the day after plating) to the non- permissive temperature for 7 days then shifted back to the permissive temperature (39°C to 33°C). Colonies were stained after two weeks (33.0°C plates) or three weeks (39.5°C and 39.5°C to 33.0°C plates) with methylene blue and counted.
Chapter 5.
146
Figure 5.3: Ability of J/1135 + 5041 cell lines to grow at the permissive temperature after shift up to the non-permissive temperature.
A. É?/1135 + 5041 cell line 1 10^ - "o 10^ - Days B. d i m s + 5041 cell line 5 lO^- 1 ‘o
I
10^“ Days 400 300 - 2 0 0-':jUI
y y (n 0\ L) Growth conditions 400 300 H 2001 1 0 0- Growth conditionsThe indicated cell lines were assayed for their ability to grow at the permissive temperature following incubation at the non-permissive temperature for 7 days. The line graphs indicate the number of cells obtained on day 1, day 4, day 7, day 10 and day 14 following the incubation of the cells (10,000 seeded per 10cm plate) at either the permissive temperature (33,0°C; — □— ), the non-permissive temperature
(39.5°C ; -o- ) or cells which were shifted (the day after plating) to the non-permissive temperature
for 7 days then shifted back to the permissive temperature ( - - - o - - ). The bar graphs represent the number of colonies obtained following the incubation of the same cell lines (1000 cells seeded per 6cm plate) after incubation at either the permissive temperature (33°C), the non-permissive temperature (39°C) or that were shifted (the day after plating) to the non-permissive temperature for 7 days then shifted back to the permissive temperature (39°C to 33°C). Colonies were stained after two weeks with methylene blue and counted.
arrested in each case, however a varying proportion of the cells, in each cell line, appeared to be able to overcome this growth arrest upon shift back to the permissive temperature. The number of cells able to overcome the growth arrest at 39.5°C or when returned to 33.0°C was least in ts\ 135+5041 cell line 10 (Fig. 5.5C) and r^l 135+5031
Chapter 5.
Figure 5.4: Ability of <//1135 + 5031 cell lines to grow at the permissive temperature after shift up to the non-permissive temperature.
A. J/1135 + 5031 cell line 7 XT' 8 %
i
Days B. <//1135 + 5031 cell line 11 107- "oI
I04- 400 300 - 2 0 0- ^ 1 0 0- Days Growth conditions 400 3 0 0 - 2 0 0- 1 0 0 - Growth conditionsThe indicated cell lines were assayed for their ability to grow at the permissive temperature following incubation at the non-permissive temperature for 7 days. The line graphs indicate the number of cells obtained on day 1, day 4, day 7, day 10 and day 14 following the incubation of the cells (10,000 seeded per 10cm plate) at either the permissive temperature (33.0°C; — □— ), the non-permissive temperature
(39.5°C ; -0- ) or cells which were shifted (the day after plating) to the non-permissive temperature
for 7 days then shifted back to the permissive temperature ( - - - o - - ). The bar graphs represent the number of colonies obtained following the incubation of the same cell lines (1000 cells seeded per 6cm plate) after incubation at either the permissive temperature (33°C), the non-permissive temperature (39°C) or that were shifted (the day after plating) to the non-permissive temperature for 7 days then shifted back to the permissive temperature (39°C to 33°C). Colonies were stained after two weeks with methylene blue and counted.
cell line 1 (Fig.5.6A). The cell number assays indicated that in each case the growth rate of this proportion of cells was similar either at 39.5°C or when returned to 33.0°C. Therefore, this may represent the sub-population of cells which failed to growth arrest at 39.5°C and not cells which had growth arrested at 39.5°C and subsequently started to
Chapter 5.
148
Figure 5.5: Ability of ^51135 + 5041 cell lines to grow at the permissive temperature following growth arrest at the non-permissive temperature.
A. /sll35 + 5041 cell line 6 8 r-o Z ,4 - 8 Days B. tsll35 + 5041 cell line 7
I
2 8 a Days C. /sll35 + 5041 cell line 10 10® -I
■or*" 8 a t - 200 y y su m as u rn m m o m Growth conditions y m y suO n U en m en o m Growth conditions Days y y s u m On U en m o en Growth conditionsThe indicated cell lines were assayed for their ability to grow at the permissive temperature after growth arrest resulting from incubation at the non-permissive temperature for 7 days. The line graphs indicate the number of cells obtained on day 1, day 4, day 7, day 10, day 14, day 17 and day 21 following the incubation of the cells (10,000 seeded per 10cm plate) at either the permissive temperature
(33.0°C; — o— ), the non-permissive temperature (39.5°C; o- - ) or cells which were shifted (the day after plating) to the non-permissive temperature for 7 days then shifted back to the permissive temper ature ( ---0-- ). The bar graphs represent the number of colonies obtained following the incubation of
the same cell lines (1000 cells seeded per 6cm plate) after incubation at either the permissive temperature (33°C), the non-permissive temperature (39°C) or that were shifted (the day after plating) to the non- permissive temperature for 7 days then shifted back to the permissive temperature (39°C to 33°C). Colonies were stained after two weeks (33.0°C plates) or three weeks (39.5°C and 39.5°C to 33.0°C plates) with methylene blue and counted.
Chapter 5.
Figure 5.6: Ability of ^sll35 + 5031 cell lines to grow at the permissive temperature following growth arrest at the non-permissive temperature.
A. tsll35 + 5031 cell line 1 *o I 105- z Days B. /sll35 + 5031 cell line 7 8 *0 2 a Days C. /sll35 + 5031 cell line 17 8 z o • 8 Growth conditions Growth conditions Days 200 - 00 - Growth conditions
The indicated cell lines were assayed for their ability to grow at the permissive temperature after growth arrest resulting from incubation at the non-permissive temperature for 7 days. The line graphs indicate the number of cells obtained on day 1, day 4, day 7, day 10, day 14, day 17 and day 21 following the incubation of the cells (10,000 seeded per 10cm plate) at either the permissive temperature
(33.0°C; — o— ), the non-permissive temperature (39.5°C; ) or cells which were shifted (the day
after plating) to the non-permissive temperature for 7 days then shifted back to the permissive temper ature ( ---0-- ). The bar graphs represent the number of colonies obtained following the incubation of
the same cell lines (1000 cells seeded per 6cm plate) after incubation at either the permissive temperature (33°C), the non-permissive temperature (39°C) or that were shifted (the day after plating) to the non- permissive temperature for 7 days then shifted back to the permissive temperature (39°C to 33°C). Colonies were stained after two weeks (33.0°C plates) or three weeks (39.5°C and 39.5°C to 33.0°C plates) with methylene blue and counted.
Chapter 5._________________________________________________ 150
proliferate when returned to 33.0°C. These cells appeared to have grown irrespective of the presence of the r^l 135 T antigen. This also appeared to be the case with tsA5S cell line 17 (Fig. 5.2C) and tsal4 (Fig. 5. IB). Both these cell lines grew similarly following 7 days at 39.5°C whether they were held at 39.5°C or returned to 33.0°C. Only with rjA58 cell line 16 (Fig. 5.2B) and tsaS (Fig. 5.1 A) did the proportion of cells which failed to arrest following 7 days at 39.5°C grow more rapidly when they were returned to 33.0°C, suggesting that the tsA5S T antigen resumed a role in the growth of this sub population of cells.
It is clear from this data that the presence of the functions carried by the 5041 or 5031 T antigen molecule at the non-permissive temperature was insufficient to prevent the m ajority of cells from becom ing irreversibly growth arrested in the ^51135+5041/5031 cell lines. The variable proportion of cells which were somehow able to overcome this growth arrest did not appear to be influenced by the réintroduction of the functions carried by the rjl 135 molecule when the cells were returned to the permissive temperature. Alternatively the r^l 135 protein may not have been sufficiently inactivated in this sub-population of cells when they were initially shifted to the non- permissive temperature.
5.2.2: Complementation of the growth defect of the conditional cell lines by the introduction of wild type or d/1135 T antigen.
I made several attempts to complement the growth defect of the conditionally immortal cell lines (both tsA5^ and r.sl 135+5041/5031) which I had isolated. The conditional cell lines were transfected with recombinants expressing either wild type (plasmid SE), d/1135 (d/1135) mutant or no (pKS) T antigen along with a recombinant plasmid expressing hygromycin-B resistance. Transfected cells were selected in hygromycin-B at either the permissive (33.0°C) or non-permissive (39.5°C) temperature. After 2-3 weeks representative colonies were isolated at the permissive temperature, the plates stained and the colonies counted. The results from three experiments are shown in Table 5.1. The number of colonies obtained at 39.5°C and 33.0°C is given along with the percentage number of colonies obtained at 39.5°C compared to 33.0°C (percentage complementation). Where the experiment was carried out more than once the average percentage complementation is shown in the right hand column. I attempted to complement the growth defect in the same cell lines used to determine the irreversiblity of the growth arrest (six tsA5S cell lines including tsaS and tsal4, three r^l 135+5041 cell lines and three f5l 135+5031 cell lines).
Of these cell lines only the tsal4 cell line was directly complemented by wild